1. Field of the Invention
The present invention relates to compositions and methods for the utilization of dynorphins for mediating physiological effects that may not be mediated entirely through the kappa opioid receptor, such as biphasic antinociception effects, motor effects, immunomodulation, inflammation response and modulation on respiration and temperature control. The present invention also relates to agonists and antagonists of dynorphins that can be found using a binding assay.
2. Reported Developments
Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and can also induce physical dependence (Koob et al., Trends Neurosci. 15:186, 1992). The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal and immune functions (Olson et al., Peptides 10:1253, 1989). Opioids exert their actions by binding to specific membrane-associated receptors located throughout the central and peripheral nervous system (Pert et al., Science 179:1011, 1973). The endogenous ligands of these opioid receptors have been identified as a family of more than 20 opioid peptides that derive from the three precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin (Hughes et al., Nature 258:577, 1975; Akil, et al., Annu. Rev. Neurosci 7:223, 1984). Although the opioid peptides belong to a class of molecules distinct from the opioid alkaloids, they share common structural features including a positive charge juxtaposed with an aromatic ring that is required for interaction with the receptor (Bradbury et al., Nature 260:165, 1976).
Pharmacological studies have suggested that there are numerous classes of opioid receptors, including those designated .delta., .kappa., and .mu. (Simon, Medicinal Res. Rev. 11:357, 1991; Lutz et al., J. Receptor Res. 12:267-286, 1992). The classes differ in their affinity for various opioid ligands and in their cellular distribution. The different classes of opioid receptors are believed to serve different physiological functions (Olson et al., Peptides 10:1253, 1989; Simon, Medicinal Res. Rev. 11:357, 1991; Lutz and Pfister, J. Receptor Res. 12:267-286, 1992). However, there is substantial overlap of function as well as of distribution. Biochemical characterization of opioid receptors from many groups reports a molecular mass of 60,000 Da for all three subtypes, suggesting that they could be related molecules (Loh et al., Annu. Rev. Pharmocol. Toxicol 30:123, 1990). Moreover, the similarity between the three receptor subtypes is supported by the isolation of (i) anti-idiotypic monoclonal antibodies competing with both .mu. and .delta. ligands but not competing with .kappa. ligands ((Gramsch et al., J. Biol. Chem. 263:5853, 1988; Coscia et al., 1991) and (ii) a monoclonal antibody raised against the purified .mu. receptor that interacts with both .mu. and .kappa. receptors (Bero et al., Mol. Pharmacol. 34:614, 1988).
Morphine interacts principally with .mu. receptors and peripheral administration of this opioid induce release of enkephalins. The .delta. receptors bind with the greatest affinity to enkephalins and have more discrete distribution in the brain than either .mu. or .kappa. receptors, with high concentrations in the basal ganglia and limbic regions. Thus, enkephalins may mediate part of the physiological response to morphine, presumably by interacting with .delta. receptors. Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase K+ conductance, and inactivate Ca.sup.2+ channels through a pertussis toxin-sensitive mechanism (Puttfarcken et al., Mol. Pharmacol. 33:520, 1988; Attali et al., J. Neurochem. 52:360, 1989; Hsia et al., J. Biol. Chem. 259:1086, 1984). These results and others suggest that opioid receptors belong to the large family of cell surface receptors that signal through G proteins (Di Chiara et al., Trends Pharmacol. Sci. 13:185-193, 1992; Loh et al., Annu. Rev. Pharmocol. Toxicol. 30:123, 1990).
Many cell surface receptor/transmembrane systems consist of at least three membrane-bound polypeptide components: (a) a cell-surface receptor; (b) an effector, such as an ion channel or the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding regulatory polypeptide or G protein, that is coupled to both the receptor and its effector.
G protein-coupled receptors mediate the actions of extracellular signals as diverse as light, odorants, peptide hormones and neurotransmitters. Such receptors have been identified in organisms as evolutionarily divergent as yeast and man. Nearly all G protein-coupled receptors bear sequence similarities with one another, and it is thought that all share a similar topological motif consisting of seven hydrophobic segments that span the lipid bilayer (Dohlman et al., Annu. Rev. Biochem. 60:653-668, 1991).
G proteins consist of three tightly associated subunits, .alpha., .beta. and .gamma. (1:1:1) in order of decreasing mass. Following agonist binding to the receptor, a conformational change is transmitted to the G protein, which causes the G.alpha.-subunit to exchange a bound GDP for GTP and to dissociate from the .beta..gamma.-subunits. The GTP-bound form of the .alpha.-subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G protein molecules, and from the stimulation by G.alpha.-GTP of many catalytic cycles of the effector. All of the family of G protein-coupled receptors appear to be similar to other members of the family of G protein-coupled receptors (e.g., dopaminergic, muscalinic, serotonergic and tachykinin), and each appears to share the characteristic seven-transmembrane segment topography.
Dynorphins are a series of peptides that share sequence homology with prodynorphin (proenkephalin B). Dynorphins are known to play a role in a wide variety of physiological parameters, including pain regulation, motor activity, cardiovascular regulation, respiration, hormone balance and the response to shock or stress. They frequently modulate the activity of other opioids, rather than having direct effects themselves. Thus, they are not analgesic in brain, yet antagonize opioid analgesia in naive animals and potentiate it in opioid tolerant animals.
Better understanding of the opioid system in analgesia will help to design more specific therapeutic drugs. In the screening process of drugs the principle operation typically is: natural agonists and antagonists bind to cell-surface receptors and channels to produce physiological effects; certain other molecules bind to receptors and channels; therefore, certain other molecules may produce physiological effects and act as therapeutic pharmaceutical agents. Thus, the ability of candidate drugs to bind to opioid receptors can function as an extremely effective screening criterion for the selection of pharmaceutical compositions with a desired functional efficacy.
After the cloning of all three major types of opioid receptors, mu, delta, and kappa, a novel receptor was cloned from several species by using homology screening technique (see: Bunzo et al., FEBS Lett. 347:284-288, (1994); Wick et al., Brain Res. Mol. Brain Res. L7:44, (1994); Wang et al., FEBS Lett. 348:75-79, (1994); and Lachowicz et al., J. Neurochem. 64:34-40, (1995). The amino acid sequence of this receptor is similar to those of the opioid receptors. However, whereas the three opioid receptors share about 70% amino acid sequence similarity among themselves, there is reduced homology level at about 65% between this receptor and any of the opioid receptors (See: Chen et al., FEBS Lett. 347:279-283, (1994)). This suggests that this novel receptor may be a member of the opioid receptor family, different from the other three receptors, and was thus design including XOR1, HOR-7, LC132, XOR, Hyp8-1, ROR-C and hORL-I. In vitro and in vivo assay systems have been used to find its ligands. A synthetic non-selective opioid agonist etorphine was shown to inhibit adenylyl cyclase in CHO-K1 cells transfected with this receptor clone, and the synthetic compounds diprenorphine and naloxone antagonized the inhibitory action of etorphine (see: Mollereau et al., FEBS Lett. 341:33-38, (1994)). However, since no endogenous (naturally existing in the body) ligands have been found for this novel receptor, it remains an "orphan" receptor.
To identify endogenous ligands for an orphan receptor, one could perform receptor binding with radiolabeled compounds. This approach has been used for the identification of 5HT.sub.1A receptor (see for example, Fargin et al., Nature 335:358-360 (1988). However, this approach is limited in its scope, since many endogenous ligands are not available in radiolabeled form. An alternative approach is to use a functional assay, in which the orphan receptor is expressed in cells and a measurable cell function is used as a readout of receptor activation, such as changes in second messenger levels or membrane currents. In this way compounds can be tested in unlabeled form and, if a proper cellular function is chosen that the orphan receptor does couple to, there is an opportunity to identify the endogenous ligands.
Xenopus oocytes have been used in many functional studies for membrane receptors and ion channels (for example by: Dascal, N. CRC Crit. Rev. Biochem. 22:317-387, (1987); and Snutch, T. P. Trends Neurosci. 11:250-256, (1988). In particular, opioid receptors have been shown to couple to a cloned G protein-activated K+ (channel (KGA) in oocytes (see for example: Dascal et al., Proc. Natl. Acad. Sci. U.S.A. 90:10235-10239, (1993) and Kavoor et al., J. Biol. Chem. 270:589-595, (1995). Because of the high degree of homology of this orphan receptor with the opioid receptors, it may also be capable of functionally coupling to KGA in Xenopus oocytes, thus constituting an assay system for identifying endogenous ligands that can activate this receptor. I took such an approach, using an opioid receptor-like orphan receptor (XOR1) cloned from rat brain (see Chen et al., FEBS Lett. 347:279-283, (1994) for oocyte expression.